This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The discovery of highly charged membrane penetrating peptides two decades ago has changed the perception that the plasma membrane is an impermeable barrier. This presented an important challenge for physical organic chemists: how does the interplay between electrostatics and vdW forces is modulated during uptake and permeation? Which structural and dynamical features of the cell penetrating peptides (CPPs), and of the lipid and solvent molecules, alter to allow a hydrophilic peptide to cross a hydrophobic core of membrane? Furthermore, numerous reports documented the capability of CPPs to transport hydrophilic cargo of varying sizes across membranes. CPPs are therefore being investigated for a variety of applications, including for the development of protein-based vaccines, the study of apoptosis and cell proliferation, transplantation of Islet cells, treatment of oxidative stress disorders, immunotherapy of tumors, and regulation of inflammation. Delivery of nucleic acids, antibodies, and imaging agents are some of the other applications of CPPs. It is apparent that physicochemical data on CPP-membrane interactions is vital for the design of novel and more effective vectors. However, in contrast to the accumulated biological and biophysical macroscopic data, information on the basic chemistry of CPP-membrane interactions is scarce. As a result, atomic-level understanding of how CPPs bind to and subsequently cross lipid bilayers is currently limited. In order to fill this void, we devised a research program that aims at characterizing the structural and thermodynamic principles underlying CPP uptake and membrane permeation. Our research will use first-principles computations, based around molecular dynamics simulations and free energy calculations, to unravel the roles of cationic charge content and amphiphiliciy of CPPs, and of membrane potential and counterions. The study will be carried out in three stages. In the first stage of the project period, we will probe which characteristics of CPPs are responsible for membrane uptake. To this end, we will compare the bilayer binding properties of arginine-rich, lysine-rich and amphipatic peptides. The second stage will focus on deciphering the mechanism of membrane translocation, with emphasis on the roles of membrane potential and counterions. The last stage concerns cargo delivery. The results will establish the physical and chemical principles of membrane uptake and permeation of CPPs by delineating the structural and energetic determinants of CPP-membrane interactions, thus laying a solid foundation for future applications-oriented studies.